| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Experimental Studies |
1 From the Department of Radiology, Hôpital Universitaire de Genève, Rue Micheli-du-Crest 24, Bâtiment C, Room 6-795, 1211 Geneva 14, Switzerland (C.M.P., X.M., F.T., J.P.V.), Pharmacy Section, Université de Lausanne, Switzerland (C.P., J.M.); Bracco Research, Geneva, Switzerland (S.P.); and Bracco Research, Milan, Italy (V.L.). Received June 10, 2002; revision requested August 20; final revision received November 26; accepted January 28, 2003. C.M.P. supported by Fond National Suisse de la Recherche Scientifique grants 3200-063619.00 and 3200-100868. Address correspondence to C.M.P. (e-mail: catherine.pastor@hcuge.ch).
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
|---|
MATERIALS AND METHODS: The authors studied both contrast agents in isolated perfused rat livers by measuring the magnetic resonance (MR) signal intensity (SI) in 12 rats, as well as the gadolinium concentrations in hepatic tissues in 42 rats. The intrahepatic transport of Gd-BOPTA was investigated with pharmacologic antagonism by using bromosulfophthalein. MR imaging was performed at 1.5 T with a fast gradient-echo T1-weighted MR sequence.
RESULTS: The hepatic kinetics based on the MR SI measured over time showed a rapid steady state during Gd-DTPA perfusion, while the SI continuously increased during the 30-minute Gd-BOPTA perfusion period. The pharmacokinetic modeling indicated that the half-lives of Gd-DTPA entry and exit were identical (mean, 1.3 minutes ± 0.9 [standard error of mean]) and shorter than those observed with Gd-BOPTA (P < .001). The uptake of Gd-BOPTA was faster (mean half-life, 4.8 minutes ± 0.3) than the washout (mean half-life, 17.5 minutes ± 2.8) (P = .001). The combined perfusion of bromosulfophthalein and Gd-BOPTA decreased the SI enhancement in comparison with the perfusion of Gd-BOPTA alone (mean, 0.56 ± 0.03 vs 2.54 ± 0.39, P < .001). The entry and exit kinetic parameters obtained during the perfusion of Gd-BOPTA plus bromosulfophthalein were identical and comparable to those obtained during Gd-DTPA perfusion (P = .95). Acute bile duct ligation did not interfere with the uptake of Gd-BOPTA in hepatocytes, but it slowed down the excretion by approximately 50%. Measurements of gadolinium concentrations in hepatic tissues confirmed these findings.
CONCLUSION: In the liver, the hepatospecific contrast agent Gd-BOPTA enters into hepatocytes likely through the organic anion transporting peptide 1.
© RSNA, 2003
Index terms: Animals • Contrast media, experimental studies • Experimental study • Gadolinium • Liver, MR, 76.121412, 76.121416, 76.12143 • Magnetic resonance (MR), contrast media, 76.12143 • Radiobiology, cell and tissue studies
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
|---|
Different categories of contrast agents, such as nonspecific extracellular gadolinium chelates, reticuloendothelial system–specific agents, and hepatocyte-specific agents, are described (2). In clinical practice, the most commonly used contrast agents are the nonspecific extracellular gadolinium chelates because they are inexpensive, safe, and well tolerated by patients, and they can enable detection and characterization of a wide range of hepatic diseases. Reticuloendothelial system–specific contrast agents (also called superparamagnetic iron oxides) are selectively taken up by reticuloendothelial system cells. Lesions that contain negligible or few reticuloendothelial system cells remain largely hyperintense, while signal intensity (SI) in surrounding normal liver is decreased as the result of the contrast agent uptake. Hepatic metastases from colon cancer are well detected with superparamagnetic iron oxides (3,4). Finally, hepatocyte-specific agents undergo uptake by hepatocytes and are partially eliminated into the bile. This category of agents enhances the SI on T1-weighted images in tissues that possess specific transporters. Normal livers and focal hepatic lesions containing hepatocytes take up these agents in contrast to lesions that do not contain hepatocytes (5,6).
In experimental studies, most findings concerning the hepatic pharmacokinetics of these contrast agents have been obtained in vivo following a systemic injection. The intravenous injection of the extracellular gadolinium chelate gadopentetate dimeglumine (Gd-DTPA, Magnevist; Schering, Berlin, Germany) is followed by a rapid decline of the plasma concentrations and a rapid distribution into the vascular compartment (7). In the liver, the agent diffuses exclusively in the extracellular space. The hepatocyte uptake and bile excretion are negligible, and the overall body excretion of the drug occurs by means of glomerular filtration.
In contrast, following an intravenous injection, gadobenate dimeglumine (Gd-BOPTA, MultiHance; Bracco Research, Geneva, Switzerland) distributes into the extracellular space and then enters into hepatocytes (8). This contrast agent is highly water soluble, exhibits weak plasma protein binding (<5%) (9), and is not metabolized (10). However, the weak binding to proteins is efficient enough to increase MR SI in plasma and tissues, as already published by Cavagna et al (9). It is eliminated via both the renal and biliary routes. The hepatic transport of Gd-BOPTA has been mainly studied in vivo by pharmacologic antagonists (11–13), and a single study found that Gd-BOPTA enters without transporter in rat basolateral (or sinusoidal) hepatocytes plasma membrane vesicles (14). Thus, no study clearly defined the hepatic transport of Gd-BOPTA.
The aim of our study was to compare in the entire liver, the hepatic kinetics of Gd-BOPTA and Gd-DTPA and to evaluate the hepatic transport of Gd-BOPTA.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
|---|
Liver Perfusion
All livers were perfused in situ as previously described (15). Briefly, the abdominal cavity was opened and the portal vein was cannulated and secured. A 16-gauge catheter (outer diameter, 1.8 mm) was introduced into the portal vein up to 2–3 mm from the liver. A ligature was placed around the inferior vena cava above the left renal vein. After the cannulation of the portal vein, the abdominal vena cava was transected and the Krebs-Henseleit-bicarbonate (KHB) solution (made in our laboratory) was pumped without delay into the portal vein. The flow rate was slowly increased over 1 minute up to 35 mL/min (increase during 1 minute and then constant flow rate 35 mL/min).
In a second step, the chest was opened and a second cannula (14-gauge) inserted through the right atrium into the thoracic inferior vena cava and secured with a ligature. Finally, the ligature around the abdominal inferior vena cava was tightened. The KHB solution was then perfused through the portal vein to the liver and eliminated by the catheter placed in the thoracic inferior vena cava. With this technique, rats die as soon as the catheter is introduced in the portal vein (hemorrhagic shock). Then, it is possible to explant the livers or leave them in the carcass, as done in our study. At the end of the surgery, four rats had an acute bile duct ligation (two ligatures tightened at the hilum). The bile duct was then resected between the two ligatures.
Liver Perfusion in the MR Imaging Room
Livers in the carcass were inserted into the wrist coil and placed in a plastic box maintained at a steady temperature (37°C). The entire perfusion system consisted of reservoir, pump (Ismatec, Glattbrugg-Zürich, Switzerland), bubble trap, filter, and oxygenator (16). The livers were perfused with a KHB buffer during the entire protocol with a nonrecirculating perfusion. The perfusate was equilibrated with a mixture of 95% O2 and 5% CO2 during the protocol. Then, the plastic box was placed inside the magnet.
MR Imaging
MR imaging (1.5 T Eclipse System; Marconi Medical Systems, Cleveland, Ohio) was performed at 1.5 T. After a scout image was obtained in the sagittal plane, a transverse image was obtained by using a fast gradient-echo T1-weighted MR imaging sequence, which was preceded by a 90° saturation pulse, with the following parameters: 6.8/3 (repetition time msec/echo time msec), 90° flip angle, 256 x 256 matrix, 14-cm field of view, 0.7-cm section thickness, and one image acquired every 8 seconds. A region of interest that encompassed a hepatic lobe and excluded all large vessels such as the portal and hepatic veins was drawn on the short-axis view of the liver. For each liver, the region of interest remained constant during the entire experiment. However, the region varied among the livers. Two authors (X.M., J.P.V.) placed the regions of interest, which ranged in size from 12 to 15 mm.
Drugs
Both Gd-DTPA and Gd-BOPTA were diluted to obtain a final concentration of 0.5 mmol/L. Bromosulfophthlalein (Sigma, Buchs, Switzerland) also was diluted to obtain a final concentration of 0.5 mmol/L.
Experimental Protocol
MR SI analysis.—In protocol 1 (Fig 1), to compare the hepatic pharmacokinetics of Gd-DTPA and Gd-BOPTA in four rat livers, we perfused each liver with KHB solution plus Gd-DTPA (20 minutes), KHB solution (10 minutes, hepatic elimination of Gd-DTPA), KHB solution plus Gd-BOPTA (30 minutes), and KHB solution (30 minutes, hepatic elimination of Gd-BOPTA). In each liver, we perfused Gd-DTPA before Gd-BOPTA.
|
In protocol 3, to acutely modify bile excretion, four livers had a bile duct ligation at the time of surgery and then were perfused with Gd-DTPA and Gd-BOPTA. The final concentrations of both contrast agents and bromosulfophthalein were 0.5 mmol/L. We used 12 rats for this component of the study.
Pharmacokinetic Modeling
The MR SI of hepatic tissues was normalized to muscle tissue and submitted to compartmental analysis by using the software MicroPharm (Inserm, version 4.0, Paris, France). The best fits were obtained with a one compartment monoexponential model according to the following two equations: For perfusion,
|
|
Gadolinium Concentrations in Hepatic Tissues
To correlate the MR SI with intrahepatic contrast agent concentrations, we perfused additional livers with KHB plus Gd-BOPTA (three livers at each time point) or KHB plus Gd-DTPA (two livers at each time point) and measured gadolinium concentrations in tissues collected 0, 1, 3, 5, 15, and 30 minutes after the start of each agent. Rats were sacrificed at each time point. Perfusate samples were also collected from the portal and hepatic veins.
To study the competition between bromosulfophthalein and Gd-BOPTA for hepatic uptake, we perfused additional livers with Gd-BOPTA plus bromosulfophthalein. Concentrations of gadolinium in tissues and perfusates were measured in similar conditions (two livers at each time point). Concentrations of gadolinium in tissues and perfusates were measured by using inductively coupled plasma atomic emission spectrometry (ICP-AES, Bracco Research, Milan, Italy). The final concentrations of both contrast agents and bromosulfophthalein were 0.5 mmol/L. We used 42 rats for this component of the study.
Hepatic O2 Consumption and Portal Pressure during Perfusion of Gd-DTPA and Gd-BOPTA
Perfusate samples were collected from the inflow and outflow tubings and immediately assayed for partial pressure of oxygen by using an ABL 505 blood gases analyzer (Radiometer, Copenhagen, Denmark) that was calibrated hourly. Hepatic O2 consumption (in microliters per minute per dry gram) was calculated from the difference in O2 contents between the inflow and outflow solutions. The solubility coefficient (24 µL of O2 per milliliter buffer) was used. The portal pressure was measured at the proximal extremity of the portal catheter (Hewlett Packard, Palo Alto, Calif). We used four rats for this component of the study.
Statistical Analysis
Data are given as means ± standard errors of the means. Data were analyzed by using one-way analysis of variance. P < .05 was considered to indicate a significant difference.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
|---|
|
|
|
|
The acute bile duct ligation did not interfere with the uptake of Gd-BOPTA in hepatocytes, but it slowed down the excretion by approximately 50%. Indeed, the entry half-life was 4.2 minutes ± 1.1 in the presence of bile duct ligation and 4.8 minutes ± 0.3 in the absence of bile duct ligation (P = .831). In contrast, the exit half-lives of Gd-BOPTA were 27.1 minutes ± 5.6 in the presence of bile duct ligation and 17.5 minutes ± 2.8 in the absence of bile duct ligation (P = .02).
Hepatic Kinetics Based on Gadolinium Concentrations
During the 30-minute perfusion of Gd-BOPTA, hepatic concentrations of gadolinium exponentially increased (Fig 5). At the end of the perfusion, the ratio between tissue and portal vein gadolinium concentrations was 75%. Additionally, when livers were perfused during 30 minutes with Gd-BOPTA plus bromosulfophthalein, the gadolinium concentration in hepatic tissues was similar to that measured in livers perfused with Gd-DTPA. The mean entry half-lives were 8.4 minutes ± 1.7 (Gd-BOPTA perfusion), 1.0 minutes ± 0.4 (Gd-BOPTA plus bromosulfophthalein perfusion), and 0.6 minutes ± 0.2 (Gd-DTPA perfusion).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
|---|
Rat Liver Perfusion: An Experimental Model for the Hepatic Kinetics of Contrast Agents
The isolated perfused liver is a useful model to study the hepatic pharmacokinetics of MR imaging contrast agents because several contrast agents can be successively studied. The perfusion of the extracellular contrast agent estimates the SI increase due to the extracellular distribution volume. According to the fast distribution of Gd-DTPA in the extracellular volume, all additional changes obtained with Gd-BOPTA are related to the uptake of the contrast agent by hepatocytes. MR imaging of isolated perfused livers has been used in few studies to assess the MR imaging of paramagnetic liposomes (17), the phagocytosis of superparamagnetic particles (18), or the detection of nitrosyl-iron complexes (19).
Hepatic SI of Gd-BOPTA and Gd-DTPA
The high hepatic enhancement of Gd-BOPTA over Gd-DTPA following intravenous injection in rats has already been shown (20). Gd-BOPTA enhances the T1-weighted MR SI according to its distribution in plasma spaces (large vessels, sinusoids, and space of Disse) in a way similar to the extracellular contrast agent. In our experimental model, because the contrast agents were perfused with a KHB containing no protein, the protein binding did not interfere with relaxivity in the vascular space. This is also suggested by the fact that when the uptake of Gd-BOPTA by hepatocytes was inhibited by bromosulfophthalein, the maximal SI of the contrast agent was similar to that of Gd-DTPA. During the perfusion of Gd-BOPTA, the SI is higher than that of Gd-DTPA due to its uptake by hepatocytes and its binding to intracellular proteins (8,21). Consequently, this contrast agent is very useful for the detection and the characterization of focal lesions in human livers (5,6). Moreover, Gd-BOPTA–enhanced MR imaging has also a great potential for the visualization of hepatitis and for the assessment of liver function (22). Interestingly, none of the contrast agents modified portal pressure or hepatic metabolism. Of note, the overall biliary excretion of Gd-BOPTA is much lower in humans (2) than in rats (12).
Transport of Gd-BOPTA into Hepatocytes
The transport of contrast agents into hepatocytes has been investigated by pharmacologic studies in vivo. These studies showed that in rats infused with bromosulfophthalein over 30 minutes before a bolus injection of Gd-BOPTA, the bile excretion of the contrast agent decreased from 36% (no bromosulfophthalein) to less than 1% (pretreatment with bromosulfophthalein) (11). Hyperbilirubinemia, by competing with the contrast agent, also reduced the biliary excretion of Gd-BOPTA from 45% to 12.8% (11). In vitro studies also investigated the transport of MR imaging contrasts agents (Table 3). Because bromosulfophthalein is transported into hepatocytes by the transporter Oatp1 and is not transported by Oatp2 (26), Gd-BOPTA would also enter via the Oatp1. However, the transport of Gd-BOPTA into hepatocytes has not been fully elucidated because in rat basolateral plasma membrane vesicles, Pascolo et al (14) found no competition for hepatocyte entry between bromosulfophthalein (100 µmol/L) and Gd-BOPTA (100 µmol/L), suggesting a passive diffusion of the contrast agent. In contrast, van Montfoort et al (24) found a transporter-mediated uptake in oocytes that was inhibited by bromosulfophthalein. In this last study, the Michaelis-Menten or affinity constant for Oatp1 transporter was 3.3 mmol/L for Gd-EOB-DTPA and 1.5 µmol/L for bromosulfophthalein.
|
Gd-EOB-DTPA is another Gd chelate that enters into hepatocytes and increases the hepatic MR imaging signal (28,29). Its uptake in hepatocytes is antagonized by bilirubin and rifamycin (21). Gd-EOB-DTPA is also transported by the rat Oatp1 but not by the rat Oatp2 (26). The agent is not taken up in oocytes injected with the cRNA encoding for the human OATP (14, 24), the rat Oatp2 (24), but it is taken up in oocytes injected with the rat Oatp1 cRNA (24) and the mouse Oatp1 cRNA (30). Bromosulfophthalein inhibited the Gd-EOB-DTPA uptake by oocytes injected with the rat Oatp1 cRNA (24). Thus, great differences in hepatic uptake exist between hepatospecific contrast agents and between species.
As we anesthetized the animals with pentobarbital, it is important to know whether this drug modifies the hepatic expression of the proteins transporting Gd-BOPTA and bromosulfophthalein. A recent study shows that administration of phenobarbital (80 mg/kg during 5 days) does not modify the expression of putative transporters of the two drugs used in our study (23). More importantly, phenobarbital does not modify the hepatic uptake of bromosulfophthalein (23).
Biliary Excretion of Gd-BOPTA
After intracellular transport, bromosulfophthalein and bilirubin are irreversibly excreted from the hepatocytes to the bile by an adenosine triphosphate–dependent transporter, Mrp2. In rat canalicular hepatocyte membrane vesicles, Gd-BOPTA is also transported by Mrp2 (25). In Mrp2-deficient rats, the biliary excretion of Gd-BOPTA is 3% while the excretion is 55% in wild-type rats (12). To impair Gd-BOPTA excretion, we chose to ligate the bile duct at the time of the surgery. However, the use of livers isolated from Mrp2-deficient rats might have been another possibility to modify Gd-BOPTA bile excretion. Interestingly, the Gd-BOPTA washout was not abolished and the ligation only slowed down the excretion in comparison with normal livers. Additionally, Gd-BOPTA might return to the hepatic microcirculation through the sinusoidal transporter Oatp1, which is bidirectionnal, or through transporters of the Mrp family.
Interestingly, when Mrp2 is down regulated (in Mrp2-deficient rats or rats with a long-lasting bile duct ligation), a new transporter, Mrp3, is up regulated in the sinusoidal membrane to compensate for Mrp2 deficit and protect hepatocytes from cell injury resulting from the accumulation of harmful organic anions (31,32). Although such Mrp3 is unlikely to be synthesized after acute ligation (the ligation was performed before liver perfusion in our model), similar mechanisms may compensate for the bile obstruction.
In our experimental model, the competition studies between bromosulfophthalein and gadobenate dimeglumine show that the contrast agent is likely to be transported by transporters of the Oatp family. However, as new transporters (33) have been recently evidenced and as differences for the hepatic transport between species have been observed, further experiments must be designed for a full understanding of the hepatic transport of these hepatocyte-specific contrast agents.
Practical applications: It is important to better understand the molecular mechanisms of hepatospecific contrast agent transport to anticipate the benefit of these agents in clinical studies. Thus, changes in the expression of these transporters in cirrhotic tissues and hepatic tumors should explain the changes in the SIs observed in clinical MR imaging.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Author contributions: Guarantors of integrity of entire study, C.M.P., J.P.V.; study concepts and design, all authors; literature research, C.M.P., J.P.V.; experimental studies, C.M.P., J.P.V., X.M., C.P.; data acquisition, X.M., C.P.; data analysis/interpretation, all authors; statistical analysis, C.M.P., C.P.; manuscript preparation and definition of intellectual content, all authors; manuscript editing, C.M.P.; manuscript revision/review and final version approval, all authors
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  H. Barash, E. Gross, I. Matot, Y. Edrei, G. Tsarfaty, G. Spira, I. Vlodavsky, E. Galun, and R. Abramovitch Functional MR Imaging during Hypercapnia and Hyperoxia: Noninvasive Tool for Monitoring Changes in Liver Perfusion and Hemodynamics in a Rat Model Radiology, June 1, 2007; 243(3): 727 - 735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Planchamp, A. Hadengue, B. Stieger, J. Bourquin, A. Vonlaufen, J.-L. Frossard, R. Quadri, C. D. Becker, and C. M. Pastor Function of Both Sinusoidal and Canalicular Transporters Controls the Concentration of Organic Anions within Hepatocytes Mol. Pharmacol., April 1, 2007; 71(4): 1089 - 1097. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Libra, C. Fernetti, V. Lorusso, M. Visigalli, P. L. Anelli, F. Staud, C. Tiribelli, and L. Pascolo Molecular Determinants in the Transport of a Bile Acid-Derived Diagnostic Agent in Tumoral and Nontumoral Cell Lines of Human Liver J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 809 - 817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Grazioli, G. Morana, M. A. Kirchin, and G. Schneider Accurate Differentiation of Focal Nodular Hyperplasia from Hepatic Adenoma at Gadobenate Dimeglumine-enhanced MR Imaging: Prospective Study Radiology, July 1, 2005; 236(1): 166 - 177. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| RADIOLOGY | RADIOGRAPHICS | RSNA JOURNALS ONLINE |
), 0.5 mmol/L Gd-BOPTA (
), 0.5 mmol/L Gd-BOPTA plus 0.5 mmol/L bromosulfophthalein (
), and 0.5 mmol/L Gd-BOPTA in livers with acute bile duct ligation (
). The combined perfusion of bromosulfophthalein and Gd-BOPTA decreases the SI enhancement in comparison with the perfusion of Gd-BOPTA alone.
), and hepatic tissues (
) during the 30-minute perfusion of Gd-BOPTA (three livers at each time point). In a similar protocol, livers were perfused with Gd-BOPTA plus bromosulfophthalein (
) (two livers at each time point). When livers were perfused during 30 minutes with both bromosulfophthalein and Gd-BOPTA, the gadolinium concentration in hepatic tissues was similar to that measured in livers perfused with Gd-DTPA.